A strain of Methylorubrum sp. PAEs 4-1 and its application in the efficient degradation of long side-chain PAEs.

Abstract

This invention provides a strain of methylbacterium ( Methylorubrum sp. PAEs 4-1 and their application in the efficient degradation of long-chain PAEs belong to the field of microbial treatment technology. This methylbacterium ( Methylorubrum sp. PAEs 4-1 was deposited at the China Center for Type Culture Collection on December 31, 2025, with accession number CCTCC NO: M 20253059. The 16S rDNA sequence is shown in SEQ ID NO.1. This Methylobacterium PAEs 4-1 can efficiently degrade long side-chain PAEs.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and more particularly to a strain of methylbacterium ( Methylorubrum sp. PAEs4-1 and its application in the efficient degradation of long side-chain PAEs. Background Technology

[0002] Phthalate esters (PAEs) are a class of plasticizers widely used in industrial production. With the large-scale use and disposal of plastic products, PAEs have become ubiquitous organic pollutants in the environment. Therefore, the degradation of PAEs is particularly important for environmental remediation.

[0003] However, long-side-chain PAEs are difficult to degrade, and existing technologies for degrading long-side-chain PAEs suffer from low degradation rates and excessively long degradation cycles. Summary of the Invention

[0004] This invention proposes a strain of methylbacterium ( Methylorubrum sp. PAEs 4-1 and its application in the efficient degradation of long-side-chain PAEs can achieve a high degradation rate of long-side-chain PAEs within a shorter degradation cycle.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a strain of methylbacterium ( Methylorubrum sp. PAEs 4-1, Methylobacterium ( Methylorubrum sp. PAEs 4-1 was deposited at the China Center for Type Culture Collection on December 31, 2025, with accession number CCTCC NO: M 20253059.

[0006] In one implementation of the first aspect, the 16S rDNA sequence of the aforementioned Methylobacterium PAEs 4-1 is shown in SEQ ID NO. 1.

[0007] In a second aspect, the present invention provides the application of the methylbacterium PAEs 4-1 of the first aspect in the efficient degradation of long side-chain PAEs.

[0008] In one implementation of the second aspect, the long-side-chain PAEs include di(2-ethylhexyl) phthalate and / or di-n-octyl phthalate.

[0009] Thirdly, the present invention provides a microbial agent for degrading long-side-chain PAEs, the microbial agent comprising the Methylobacterium PAEs 4-1 provided in the first aspect.

[0010] Fourthly, the present invention provides a method for degrading long-side-chain PAEs, wherein the method involves applying the Methylobacterium PAEs 4-1 provided in the first aspect or the bacterial agent provided in the third aspect to water contaminated by the degradation of long-side-chain PAEs.

[0011] Compared with the prior art, the present invention has the following beneficial effects.

[0012] The Methylobacterium PAEs 4-1 provided by this invention can grow and reproduce using di(2-ethylhexyl) phthalate and di-n-octyl phthalate as the sole carbon and energy sources. Under pure culture conditions, this bacterium can almost completely degrade a mixture of 20 mg / L PAEs (containing 20 mg / L DEHP and DnOP respectively) in an inorganic salt medium in 7 days, with a degradation rate of over 80%. Therefore, the Methylobacterium PAEs 4-1 provided by this invention can achieve a high degradation rate of long-side-chain PAEs in a relatively short degradation cycle. Attached Figure Description

[0013] Figure 1 This is the growth morphology of Methylobacterium PAEs 4-1 provided in this application embodiment after 3 days of culture on LB medium; Figure 2 This is a scanning electron microscope image of Methylobacterium PAEs 4-1 provided in the embodiments of this application; Figure 3 This is a schematic diagram of the phylogenetic tree of 16S rDNA of Methylobacterium PAEs 4-1 provided in the embodiments of this application; Figure 4 This is a schematic diagram illustrating the degradation effect of Methylobacterium PAEs 4-1 on long-side-chain PAEs provided in the embodiments of this application. Detailed Implementation

[0014] In the specification and claims of this invention, the terms "first" and "second" are used to distinguish different objects, rather than to describe a specific order of objects.

[0015] In the embodiments of this application, "and / or" indicates a relationship between objects. For example, A and / or B can represent the following three situations: A exists alone, B exists alone, and A and B exist simultaneously.

[0016] In the embodiments of this application, the terms "exemplary" or "for example" are used to indicate that something is an example, illustration, or description. Any embodiment or design that is described as "exemplary" or "for example" in the embodiments of this application should not be construed as being more preferred or advantageous than other embodiments or design. Specifically, the use of the terms "exemplary" or "for example" is intended to present the relevant concepts in a specific manner.

[0017] Understandably, among the many PAEs, long-chain PAEs, due to their longer alkyl side chains, greater steric hindrance, and stronger hydrophobicity, are more persistent in the environment. Traditional physicochemical treatment methods are costly and prone to secondary pollution. Therefore, biodegradation technology, especially the use of highly efficient degrading microorganisms for environmental remediation, has become a current research focus and the most promising solution. The most common long-chain PAEs currently include di(2-ethylhexyl) phthalate (DEHP) and di-n-octyl phthalate (DnOP).

[0018] While biodegradation technology for environmental remediation (such as using degrading strains to degrade PAEs) is currently the most promising solution, most existing degrading strains still face several bottlenecks in practical applications: First, their degradation efficiency for long-chain PAEs (such as DEHP) is generally low and the degradation cycle is long, making it difficult to meet the needs of rapid on-site remediation; second, many strains are sensitive to changes in environmental conditions (such as pH, temperature, salinity, and coexisting pollutants), and their adaptability and stability are insufficient.

[0019] To address the issues of low degradation rates and excessively long degradation cycles in existing technologies for the degradation of long-side-chain PAEs, this application provides a strain of methylbacterium (…). Methylorubrum sp. PAEs4-1 and its application in the efficient degradation of long-side-chain PAEs can achieve a high degradation rate of long-side-chain PAEs within a shorter degradation cycle.

[0020] Example 1: This example describes a strain of methylbacterium ( Methylorubrum sp. PAEs 4-1, Methylobacterium ( Methylorubrum sp. PAEs 4-1 was deposited at the China Center for Type Culture Collection on December 31, 2025. The accession number for Methylbacterium PAEs 4-1 is CCTCC NO: M 20253059, and the deposit address is Wuhan University, Wuhan, China.

[0021] The 16S rDNA sequence of the above-mentioned Methylobacterium PAEs 4-1 is shown in SEQ ID NO.1.

[0022] SEQ ID NO.1: The sequence similarity of the aforementioned Methylobacterium PAEs 4-1 was compared with existing PAEs-degrading strains. The comparison showed that the 16S rDNA sequence of the aforementioned Methylobacterium PAEs 4-1 is similar to that of known degrading strains. Paenarthrobacter The sp. strainPH1 has a sequence similarity of 79.04% (<98.65%), which indicates it is a potential new species.

[0023] As is generally known in the field, the "Preliminary Identification of Bacterial Species Based on 16S rDNA Gene and Genomic Sequence" clearly states that when the similarity between the 16S rDNA gene sequences of two strains is less than 98.65%, they can be determined to belong to different species. Therefore, the isolation and identification process of the aforementioned Methylobacterium PAEs 4-1 can be understood.

[0024] The isolation and identification process of the above-mentioned Methylobacterium PAEs 4-1 is given below.

[0025] 1. Culture medium formulation Inorganic salt medium (MSM): (NH4)2SO4: 1.5 g / L; KH2PO4: 0.5 g / L; K2HPO4·3H2O: 1.91 g / L; NaCl: 0.5 g / L; MgSO4·7H2O: 0.2 g / L; adjust the final pH of the inorganic salt medium to 7.0; add 1.5% (w / v) agar powder to the MSM solid medium.

[0026] LB liquid medium: Yeast extract: 5.0g; Tryptone: 10.0g; Sodium chloride (NaCl): 10.0g; Add ultrapure water to 1L, adjust pH=7.0, and sterilize at 121℃ for 20 minutes; LB solid medium is prepared by adding 1.5% (w / v) agar powder.

[0027] 2. Separation and Identification 2.1 Isolation and Culture of Strains Weigh approximately 5g of the test soil (PAEs-contaminated soil from Qixia District, Nanjing City) and add it to a 250mL Erlenmeyer flask. Add 100mL of ultrapure water and place the flask in a shaker at 30℃ and 150rpm in the dark for 8 hours. After 8 hours of shaking culture, remove the flask and let it stand for 2 hours. The supernatant obtained after standing is the enriched initial indigenous microorganism.

[0028] Take 5 mL of the supernatant and transfer it to 95 mL of inorganic salt medium containing PAEs (5 mg / L DEHP, 5 mg / L DnOP). Incubate at 30 ℃ and 150 rpm for 5 days with shaking. Then, continuously enrich the medium with 5 mL of the inoculum, subculturing 5 times, increasing the PAE concentration in the medium to 80 mg / L accordingly (i.e., containing 80 mg / L DEHP and 80 mg / L DnOP; PAE concentration in the medium after the first subculture is 5 mg / L; after the second subculture, it is 10 mg / L; after the third subculture, it is 20 mg / L; after the fourth subculture, it is 40 mg / L; and after the fifth subculture, it is 80 mg / L). Then, dilute the culture medium obtained after the above 5 subcultures by 10%. 3 ~10 4 The culture was spread onto MSM solid medium containing 20 mg / L PAEs contaminants and incubated upside down at 30°C for 1–3 days. After single colonies grew on the plates, each colony was picked, streaked multiple times for purification, and a single bacterial strain was isolated and labeled PAEs 4-1. The strain was then inoculated onto LB solid medium and incubated upside down at 30°C for 5 days, and its colony morphology was observed.

[0029] Observation shows that the colonies of the above-mentioned strains are round, pinkish, opaque, slightly raised, with neat edges and no halo. Specific morphology is as follows: Figure 1 As shown.

[0030] 2.2 Strain Identification Scanning electron microscopy (SEM) identification: The purified strain PAEs 4-1 was inoculated into LB liquid medium and activated overnight. 1 mL of the bacterial culture was then centrifuged at 8000 rpm for 3-5 min, the supernatant was discarded, and the bacteria were washed three times with MSM. 1 mL of 2.5% (v / v) glutaraldehyde was added to the harvested bacterial precipitate and mixed thoroughly. The mixture was incubated overnight at 4°C. The glutaraldehyde was discarded, and the bacteria were washed three times with 0.1 M, pH 7.0 PBS for 15 min each time. The sample was fixed with 1% osmium tetroxide solution for 1-2 h. The osmium tetroxide waste solution was removed, and the sample was rinsed three times with 0.1 M, pH 7.0 PBS for 15 min each time. The sample was dehydrated with a gradient of concentrations of ethanol (30%, 50%, 70%, 80%, 90%, and 95%) for 15 min at each concentration, followed by treatment with 100% ethanol for 20 min. Finally, the sample was treated with pure acetone for 20 min.

[0031] The sample was treated with a mixture of embedding agent (epoxy resin) and acetone (V / V=1 / 1) for 1 hour; then with a mixture of embedding agent and acetone (V / V=3 / 1) for 3 hours; and finally with pure embedding agent overnight. The infiltrated sample was then embedded and heated at 70°C overnight to obtain the embedded sample. The sample was sectioned using an ultramicrotome to obtain sections of 70–90 nm. The sections were stained with lead citrate solution and 50% ethanol-based uranium acetate solution for 5–10 minutes each, and then air-dried for observation under a transmission electron microscope. Scanning electron microscopy revealed an elliptical morphology. Figure 2 .

[0032] 3. Molecular identification of the strain's 16S rDNA Total bacterial DNA was extracted, and the bacterial genome was amplified by PCR using universal primers for bacterial 16S rDNA. The PCR products were sequenced (by Shanghai Sangon Biotech), and the sequencing results were compared for homology with reported 16S rDNA sequences in GenBank. Phylogenetic analysis was then performed on relevant bacterial species, and the results are shown below. Figure 3 As shown.

[0033] Depend on Figure 3 It can be seen that the 16S rDNA gene sequence of strain PAEs 4-1 isolated and purified by the above process was compared with the 16S rDNA sequences of other registered bacterial strains using the BLAST program on the NCBI official website. The results showed that this strain is similar to Methylobacterium ( Methylorubrum The strains obtained through screening showed the highest similarity to *S.*, with a homology rate of 99%. Therefore, the strains obtained through screening were identified as *Methylobacterium* (sp.). Methylorubrum sp.), named Methylobacterium ( Methylorubrum sp.) PAEs 4-1.

[0034] After isolating the above-mentioned Methylobacterium PAEs 4-1, the verification process of the degradation effect of the above-mentioned Methylobacterium PAEs 4-1 on long side chain PAEs is as follows.

[0035] 1. Preparation of bacterial suspension Methylobacterium PAEs 4-1 was inoculated into 100 ml LB medium and cultured at 30 °C and 150 rpm for 24 h. After centrifugation at 8000 rcf for 5 min, the culture medium was washed twice with MSM, and the OD values ​​of Methylobacterium PAEs 4-1 were calculated. 600 Adjust the value to 1.0 to prepare a bacterial suspension, and store it temporarily at 4℃ for later use.

[0036] 2. Degradation performance determination of Methylobacterium PAEs 4-1 One mL of the above bacterial suspension was inoculated into 19 mL of MSM containing 20 mg / L PAEs (i.e., 20 mg / L DEHP and 20 mg / L DnOP). MSM without this bacterial suspension served as a control group. The pH was adjusted to 7.0, and three replicates were set for each group. The samples were incubated at 30°C and 150 rpm for 7 days. Samples were taken on days 1, 3, 5, and 7. 40 mL of chromatographically pure methanol was added to the collected Erlenmeyer flasks, and the mixture was sonicated in a water bath for 1 hour. After sonication, the mixture was vortexed, and the supernatant was filtered through a 0.22 μm organic phase filter membrane and transferred to a 2 mL amber liquid chromatography vial for analysis using high-performance liquid chromatography (HPLC).

[0037] Chromatographic conditions: An LC-20AT high-performance liquid chromatograph (equipped with an SPD-2A UV detector) was used. The detection time was 40 min, and the injection volume was 20 μL. The separation system used acetonitrile-water as the mobile phase with an initial flow rate of 1.0 mL / min, employing gradient elution to separate PAEs. The chromatographic column was a Φ4.6×250 mm Inertsil ODS-P HPLC column, and the column temperature was 40℃. The detection system used a UV detector in dual-wavelength detection mode at 205 nm and 225 nm.

[0038] The degradation effect of Methylobacterium PAEs 4-1 on the two mixed long-side-chain PAEs (DEHP and DnOP) obtained by the above tests is as follows: Figure 4 As shown. By Figure 4 It can be seen that the above-mentioned Methylobacterium PAEs 4-1 has a significant degradation effect on both PAEs under 7 days of shaking culture. The degradation rate of both PAEs by Methylobacterium PAEs 4-1 exceeded 50% on the first day; on the 7th day, the degradation rate of DEHP exceeded 80% and the degradation rate of DnOP exceeded 90%, indicating that the above-mentioned Methylobacterium PAEs 4-1 has a highly efficient degradation ability for long side-chain PAEs.

[0039] Example 2: This example describes the application of Methylobacterium PAEs 4-1 in Example 1 in the efficient degradation of long side-chain PAEs.

[0040] Optionally, the aforementioned long side-chain PAEs may include di(2-ethylhexyl) phthalate (DEHP), di-n-octyl phthalate (DnOP), or other side-chain PAEs. The embodiments of this application do not limit the types of the aforementioned side-chain PAEs.

[0041] Specifically, the above applications can be as follows: the bacterial suspension of the above-mentioned Methylobacterium PAEs 4-1 (the preparation process is described in the preparation section of the bacterial suspension in Example 1) can be directly inoculated into water bodies contaminated with PAEs; or the bacterial agent prepared from the above-mentioned Methylobacterium PAEs 4-1 can be inoculated into soil / water bodies contaminated with PAEs to achieve the degradation of long side-chain PAEs.

[0042] It is understood that the preparation process of the above-mentioned bacterial agent can involve activating the above-mentioned Methylobacterium PAEs 4-1 and then loading it onto an activated carbon carrier to obtain the bacterial agent. The above-mentioned activated carbon carrier can be obtained by carbonizing rice straw or corn stalks, and this embodiment is not limited thereto. The above-mentioned preparation process is a commonly used technical means in this technical field, and this embodiment will not elaborate on it here.

[0043] Example 3: This example describes a bacterial agent for degrading long side-chain PAEs, which includes Methylobacterium PAEs 4-1 from Example 1.

[0044] In one application scenario, when the above-mentioned bacterial agent is a bacterial suspension, the preparation method of the bacterial suspension is as follows.

[0045] Step 1.1: Inoculate Methylbacterium PAEs 4-1 into 100 ml LB medium and incubate at 30 ℃ and 150 rpm for 24 h. Then centrifuge at 8000 rpm for 5 min to obtain activated Methylbacterium PAEs 4-1.

[0046] Step 1.2: After washing the activated Methylobacterium PAEs 4-1 twice with MSM, the OD values ​​of the washed Methylobacterium PAEs 4-1 were... 600nm The value was adjusted to 1.0 to obtain a bacterial suspension, which was then stored at 4 ℃ for later use.

[0047] In another application scenario, when the above-mentioned bacterial agent is an immobilized bacterial agent, the preparation method of the immobilized bacterial agent is as follows.

[0048] Step 2.1: Inoculate endophytic Acinetobacter Sb 2-4 into 100 ml LB medium and incubate at 30 ℃ and 150 rpm for 24 h. Then centrifuge at 8000 rpm for 5 min to obtain activated endophytic Acinetobacter Sb 2-4.

[0049] Step 2.2: After carbonizing rice straw or corn stalks at high temperature, activated carbon carrier is obtained.

[0050] For example, the high-temperature carbonization temperature can be 800°C or 1200°C, or other values ​​within a reasonable range. This application embodiment does not limit the high-temperature carbonization temperature and carbonization time.

[0051] Step 2.3: Immobilize Methylbacterium PAEs 4-1 on an activated carbon carrier to obtain an immobilized bacterial agent of Methylbacterium PAEs 4-1.

[0052] Optionally, the viable count of Methylobacterium PAEs 4-1 loaded on the activated carbon carrier can be 2.5 × 10⁻⁶. 10 The CFU / g value can also be any other value within a reasonable range. This application does not limit the above-mentioned viable count and loading time in its embodiments.

[0053] Example 4: This example describes a method for degrading long-side-chain PAEs, in which the Methylobacterium PAEs 4-1 from Example 1 or the bacterial agent from Example 3 is applied to water contaminated with degraded long-side-chain PAEs.

[0054] For example, when the medium is water, the method can be to directly inoculate Methylbacillus PAEs 4-1 into the water contaminated with PAEs, or to inoculate a bacterial suspension / agent containing Methylbacillus PAEs 4-1 into the water contaminated with PAEs to degrade the PAEs in the water. The inoculation amount of Methylbacillus PAEs 4-1, the inoculation amount of the bacterial suspension, and the inoculation amount of the agent can all be 5%-10%; this embodiment does not limit this.

[0055] It is understood that the aforementioned water body can be water contaminated with phthalates, or it can be sewage contaminated with phthalates in a sewage treatment plant (such as sewage in a biological reactor and a secondary sedimentation tank). This application embodiment does not limit the type and scenario of the aforementioned water body.

[0056] The various embodiments in this specification are described in a progressive manner. The same or similar parts between the various embodiments can be referred to each other. Each embodiment focuses on describing the differences from other embodiments.

[0057] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of this application.

Claims

1. A strain of methylbacterium ( Methylorubrum sp. PAEs 4-1, characterized in that, The methylbacterium ( Methylorubrum sp. PAEs 4-1 was deposited at the China Center for Type Culture Collection on December 31, 2025, with accession number CCTCC NO: M 20253059.

2. The application of the Methylobacterium PAEs 4-1 as described in claim 1 in the efficient degradation of long side-chain PAEs.

3. The application as described in claim 2, characterized in that, The long-chain PAEs include di(2-ethylhexyl) phthalate and / or di-n-octyl phthalate.

4. A microbial agent for degrading long-chain PAEs, characterized in that, The bacterial agent contains the Methylobacterium PAEs 4-1 as described in claim 1.

5. A method for degrading long-side-chain PAEs, characterized in that, Apply the Methylobacterium PAEs 4-1 of claim 1 or the bacterial agent of claim 4 to water bodies contaminated by degraded long-chain PAEs.

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